| 1. |
- Duong-Thi, Minh-Dao, et al.
(författare)
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Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins
- 2016
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Ingår i: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 141:3, s. 981-988
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures.
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| 2. |
- Duong-Thi, Minh-Dao, et al.
(författare)
-
Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins
- 2016
-
Ingår i: The Analyst. - 0003-2654 .- 1364-5528. ; 141:3, s. 981-988
-
Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures.
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| 3. |
- Ohlson, Sten, et al.
(författare)
-
Direct analysis - no sample preparation - of bioavailable cortisol in human plasma by weak affinity chromatography (WAC)
- 2017
-
Ingår i: Journal of chromatography. B. - : Elsevier. - 1570-0232 .- 1873-376X. ; 1061, s. 438-444
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Tidskriftsartikel (refereegranskat)abstract
- Pre-analytical treatment of blood plasma is a time consuming and often rate limiting step in the workflow of LC/MS analysis. We present in this pilot study a new approach for quantitative LC/MS based on weak affinity chromatography (WAC) of crude plasma. The steroid hormone cortisol was selected as a clinically relevant biomarker, as it currently requires extensive pre-analytical preparation. A WAC unit with saturating, immobilized albumin as a prototypic weak binder was used in combination with an ion-funnel MS/MS detector to perform zonal affinity chromatography of cortisol directly from a plasma sample, followed by quantitative multiple reaction monitoring (MRM). This procedure also allowed us to determine the amount of bioavailable cortisol in the clinical plasma sample which is of significant therapeutic interest. This WAC-MS approach showed an excellent correlation (R-2 = 0.86 (P < 0.0001 (highly significant); n = 60) with a state-of-the-art, clinical competitive immunoassay procedure for plasma cortisol analysis. With integration of WAC into LC/MS workflow, it may be possible to both accelerate and improve assay performance by eliminating the sample extraction step. Preliminary data with other steroid hormones indicate that WAC-MS can be applied to various biomolecules using a plasma transport protein such as albumin.
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